The present disclosure relates to recombinases and methods of using homologous recombination.
In the past several years, the genome project has provided knowledge that was once thought to be nearly impossible to obtain. Thanks to this endeavor, we now have a window into the very code of life. One of the original promises of this work was that having the actual sequences of the human genome would enable the detection of differences in those sequences that lead to disease or malfunction, and may eventually lead to correction of such defects. However, because the genomes of higher organisms are both complex and large, their manipulation is not a simple matter.
While techniques such as cloning site-specific mutations using DNA restriction enzymes have been successful for manipulating DNA fragments, such techniques are not well-suited for manipulation of large DNA fragments, i.e., fragments the size of mammalian genes. One method that has been used to manipulate large DNA fragments is recombination, particularly site-specific recombination. Recombination is the exchange of DNA segments along two different strands of DNA. In site-specific recombination, DNA strand exchange occurs at a specific site, for example, as in the integration of phage lambda into the E. coli chromosome and the excision of lambda DNA from it. Site-specific recombination involves specific sequences of both the donor and target DNA segments. In the Cre-loxP and FLP-FRT systems, for example, recombination involves short (i.e., less than about 50 base pairs), inverted repeat sequences. Within these sequences, the homology between the DNA sequences is necessary for the recombination event, but not sufficient for it. Site-specific recombination requires enzymes or multi-enzyme complexes, often called recombinases. In site-specific recombination, recombinases generally cannot recombine other pairs of homologous (or nonhomologous) sequences, but act specifically on particular DNA sequences. Site-specific recombination has been proposed as one method to integrate transfected DNA at chromosomal locations having specific recognition sites. Because this approach requires the presence of specific target DNA sequence and recombinase combinations, its utility for targeting recombination events at a particular chromosomal location is limited.
Homologous recombination (or general recombination), in contrast, is defined as the exchange of homologous segments anywhere along a length of two DNA molecules. A feature of homologous recombination is that a recombinase active for homologous recombination can often use any pair of homologous sequences as substrates, although some types of sequence may be somewhat favored over others.
Several recombinases that catalyze homologous pairing and/or strand exchange in vitro have been purified and at least partially characterized, including: E. coli recA protein, the phage T4 uvsX protein, and the red protein from Ustilago maydis. Recombinases, such as the recA protein of E. coli, are proteins that promote strand pairing and exchange in such important cellular processes as the SOS repair response, DNA repair, and efficient genetic recombination in E. coli. RecA can catalyze homologous pairing of a linear duplex DNA and a homologous single strand DNA in vitro.
One drawback to the use of previously characterized recombinases such as recA is that these proteins are from prokaryotes and simple eukaryotes, and may not be applicable to recombination in higher eukaryotes such as mammals. There thus remains a need for compositions and methods for gene manipulation using homologous recombination that are suitable for use in mammalian hosts.